PASSIVE TOOLS
CUTANEOUS
ANAPHYLACTIC
IN THE STUDY
REACTIONS
OF THE STRUCTURE
AS
OF THE
IgG MOLECULE Z. OVARY Department ol Pathology. New York University School of Medicine. 550 t’irst A\enue. New York. NY 10016. U.S.A.
anaphylaxis it has been shown that the ‘ttructurL‘neccs%iry for lisatior~ the Fc fragment. Fc fragments from rabbit IgG czn Inhibit passiw cutaneous anaphylaxis in guinea pigs by competltion [or the receptor. The fixation ol’the Fc ltagment to receptors (on mastcells) was shown also by reverse pawve cutaneous anephylaxis usmg dinitrophenylated Fc f&ments and anti-dinitrop~cn}~l antibodies. Absract-
By use ofpassivecutaneous
to tissue constitutents (receptors) is on
Before sequencing of poiypeptid~ chains became possible, two methods were in use for study of the antibody molecule: digestion by proteolytic enzymes (such as papain and pepsin} to obtain smaller fragments which could be studied with lesser difficulty, and the separation of the constituent chains. Both methods were introduced by Porter (1958, 1959,1962). Using digestion by papain, the IgG molecule is split in three fragments: two identical Fab and one Fc. When these fragments were obtained from rabbit IgG it became possible to examine their functions in vitro and
different
be in~estigatcd
&I
It has been known for a long time that to provoke PCA reactions a suitable time is needed between the injection of the antibody and the challenge by the antigen (Ovary & Bier, 1953~) which we call the ‘sensitization period’. If rabbit antibody is injected into the skin of a guinea pig and after a suitable sensitization period (i.e. 2-6 hr) the antigen is injected together with a dye, a colered spot develops at the site of the antibody injection a few minutes after the ‘challenging’ antigen injection. This reaction is due to the increased permeability of the postcapillary venules. The increased permeability is produced by liberation of vasoactive substances from mast cells. When, as just described, the antibody is injected intradermally and the antigen intravenously, this is modality I of the PCA reaction, the most generally used modality (Watanabe & Ovary, 1977). Very small amounts of rabbit antibody can be detected by this method (Ovary, 1964). With small amounts of antibody. relatively large amounts of antigen are needed (for example, with 0.5 fig of rabbit antiovalbumin antibody/ml. 30 pg of ovalbumin:guinea pig are necessary to have a n~aximum reaction). Antigen excess is not in’hibitory. With Karush (Ovary & Karush. 1961), I used purified rabbit anti-p-azophenyl+lactoside (antiLAC) IgG antibody and its Fab and Fc fragments to investigate by PCA one of the biological activities of rabbit IgG antibody: the capacity to sensitize guinea pig skin for an~phylactic reactions. When purified rabbit anti-LAC was used, PCA reactions were obtained. No reactions, however, were obtained with the Fab or the Fc fragments alone even when they were injected at a concentration 25 times greater than that used to elicit PCA with intact antibody. When 50 times the concentration of the Fab fragment was mixed with the non-digested antibody. the PCA reaction was inhibited if- the sensitization period was 2 hr, but not when it was 5 hr. provided that relatively small amounts of antigen were used for challenge (i.e. 30 pg pf LAC-fibrinogen). Table 1
in viva. fn vitro it was shown that the Fab fragment
contains the antigen combining site and that it is monovalent, therefore, it does not precipitate the antigen though it combines with it. The Fc fragment has no specific antigen recognition site (Porter, 1958, 1959). The Fc fragment is much more uniform than the Fab fragment and rabbit Fc was easily crystallized. Later, when Hilschmann discovered that the polypeptide chains which make up the immunoglobulin have an N terminal variable and a C terminal constant portion (Hilschmann & Graig, 1965) and the Fc fragment contains no variable portion of the H chain (Hill et ul., 1967) it was readily understood why the Fc fragment is crystallizable. Nisonoff used pepsin to digest the IgG molecule (Nisonoff et a/., 1960) and showed that this enzyme cleaves the Fc segment into small peptides leaving a bivalent fragment F(ab’)$ undigested. This bivalent fragment has two combmmg sites and precipitates the antigen. but has no Fc portion. The question was asked: do these fragments still have biological activity and if so where is the biological function located, in which of the fragments? Passive cutaneous anaphylaxis (PCA)* (Ovary, 1951. 1964) was used to investigate these questions. Advantage was taken of the fact that rabbit IgG antibody is capable of sensitizing the guinea pig for anaphylactic reactions and therefore the role of the ___.ll___l___used: p-arophmyl-l,‘-iactosidc. * Abbreviations passive cutaneous ansphyiaxis. PCA.
rabbit IgG f~dgmentscould
viva.
LAC: 751
Z. OVARY
752
Table 1. Lack of PCA reaction by papain digest fragments ot rabbit anti-LAC antibody and Inhibition of PC’A reaction by Fab (anti-LA(“) lnt~dcrn~~~l injectlon of antibody pro-
tein in 0.1 ml Anti-LAC Anti-LAC Fab Fab i-c l-c Anti-LAC Fab Anti-LAC Fab
+ +
pg,ml I’ I’ hOO 600 600 600 I.! 600 I2 600
Sensitization period (hr)
Diameter of” rcilctwn\ in mm
2 5 2 5 7 5
10 IS U 0 0 0
10 15 0 0 0 0
I7 Iti 0 0 0 0
IS 20 0 0 0 0
2
0
0
0
0
s
1’
IO
12
Ii
shows a representative experiment. When the antigen amount was increased to 500 ~(6 no inhibition occurred. Similar results were obtained with many other systems (anti-o~~a~burn~n, anti-human IgG. antidinitrophenyl antibodies and other systems). The inhibition of PCA reactions by the Fab fragment is specific: Fab fragments from anti-LAC antibody do not inhibit PCA reactions by anti-ovalbumin (Ovary & Karush. 1961). The Fab fragInent dots not sensitirc for anaphylactic reactions and it is monovalent. Does it not sensitize because it is monovalent or for some other reason? It was shown that monovalent haptcns like epsilon-DNP-lysine cannot be used to challenge for anaphylactic reactions whereas bivalent haptcns such as the alpha-epsilon-his-DNP-lysine are effective (Ovary, 1961. 1963). When hybrid antibody molecules are prepared from specific IgG antibodies according to the method of Nisonoff (Nisonoff & Palmer. 1964) the antibody molecule has only one combining site for the specific antigen in question if for hybridization normal rabbit IgG is used. Hybrid antibodies do sensitize for PCA reactions with multivaie~~t antigens (Ovary, 1965). observations (ineffectiveness of The above monovalent haptens as challenging antigens, effectiveness of hybrid antibodies) lead me to the the
‘bridging hypothesis’ (Ovary, 1961. 1965); namely that two different antibody molecules ‘fixed’ to the receptors of mast cells must be ‘bridged’ by a piuri~dient antigen to challenge anaphyiactic reactions. It did not seem probable that the ineffectiveness ot the Fab antibody fragment for sensitization wasdue to its monovalency because plurivalent antigens are capable of bridging two Fab fragments and also because the inhibition produced by Fab fragments of anaphyiactic reactions by the intact antibody is of short duration (‘Fable I). To investigate this further we used bivalent rabbit antibody fragments. namely the F(ab’), fragment. F(ab’)2 fragments obtained from purified rabbit antibody by the method of Nisonoff (Nisonoff c’t u/., 1960) were also unable to sensitize guinea pig skin for PCA reactions (Ovary & Taranta, 1963; Ovary, 1965). When a great excess of F(ah’f2 fragment (more than 25 times on a weight basis) was mixed with the intact antibody, inhibition of PCA reactions occurred, provided that the F(ab ), fragment had the same specificity as the intact anti-body. that the sensitization period was kept short (e.g. 2 hr) and that the antigen amount used for challenge was not too excessive (at maximum, 100 itg). In other words. the F(ab’): fragment behaved similarly to the Fab fragment. Therefore. the inability of the Fab antibody fragment to sensitize guinea pig skin was not due to the fact that it was monovalent. but to something else. The results oi‘ these experiments indicated that the structure necessary to react with the tissue receptors. i.e. the receptors on the mast ce!ls, is not on the t;ab or the F(ab’)2 fragments. It was already known that when purified antibody was mixed with antibody of unrelated speciticity no PCA reactions could be obtained. To obtain such an inhibition it was necessary to use at least 15 times more unrelated antibody than the antibody used for PCA reactions (Ovary t’t (11.. 1963). This observation was 111 accordance with previous observations which showed that when antibody is diiutod in normal rabbit serum instead of physiological saline. inhibition results (Ovary di Bier. 1963h). The inhibition of PCA reactions with unrelated antibody was, however. different from the inhibition obtained by the Fab or the F(ab’), fragments because it could not he overcome Gy prolongation of the srnaltization period or by increasing the amount of the challcnglng
Passive Cutaneous
Table
3. Reverse PCA
Anaphylactic
reactions with rabbit IgG digest fragments
0.1 ml intradcrmally
antibody
12 12 50 50 5 5 5 5
and its papain
Diameter
f-e protein:ml
Anti-LAC Anti-DNP Fab from antl-LAC Fab from anti-DNP Fc from anti-LAC kc from anti-DNP DNP, ,Fc from anti-DNP DNP,Fc from anti-DNP
753
Keactlons
of
reactions” mm 0 0 0 0 0 0 0 17
0 0 0 0 0 0 0 1x
0 0 0 0 0 0 0 IH
0 0 0 0 0 0 0 16
Sensitllation period 4 hr. Challenge by 400 l(g rabbit-anti-DNP. “ Eberv vertical column shows realts obtalned on the same guinea pig.
antigen. Similar inhibition was obtained using normal rabbit gamma globulin. When an Fc fragment was mixed with the sensitizing antibody instead of an unrelated antibody or normal IgG, the same inhibition was obtained, even if the Fc fragment was obtained from the same antibody preparation which was used to provoke PCA reactions. The competition for the mast cell ‘Fc receptor’ between the sensitizing antibody (the intact anti-DNP antibody molecule for example) and the unrelated antibody (anti-ovalbumin antibody) or the normal IgG molecule or the Fc fragment explains satisfactorily this type of inhibition of PCA reactions. The concluding evidence that the Fc fragment has a structure which is readily recognized by mast cell receptors was given by reverse PCA reactions (Van den Ende. 1940: Bier & Siqueira, 1956: Ovary, 1960). If intact rabbit IgG is injected into guinea pig skin, after a suitable sensitization period a reaction can be provoked if any antibody against the rabbit IgG molecule is injected intravenously. To visualize the reaction a dye is injected together with the challenging antibody. The origin of the antibody with which reverse PCA reactions can be provoked is unimportant. It can be from the horse. chicken, goat or another species which does not sensitize the guinea pig for anaphylactic reactions, It can even be a guinea pig IgGl antibody (which sensitizes the guinea pig) (Ovary, 1960). The specificity can be directed against antigenic determinants specific for the Fc fragment, the Fab fragment or for the light chains (Ovary et al., 1970). A similar reaction can be provoked when, instead of the intact IgG molecule. an Fc fragment is used. In this case. however, a prerequisite is that the antibody used for challenge must react with Fc determinants. When Fab or F(ab’), fragments are used for sensitization no reverse PCA reaction can be provoked even when care is taken that the antibody used for challenge has specificities for the Fab determinants or for the L chams. The most convincing demonstration that the Fc fragment has structures for ‘fixation’ to receptors on mast cells was made using the Fc fragment prepared
from a sample of purified rabbit anti-DNP antibody. The Fc fragment obtained by Porter’s method was coupled to dinitrophenyl by the method of Eisen (Eisen et al., 1954; Ovary & Benacerraf, 1963). Care was taken to couple less than 9 DNP groups per molecule of Fc fragment as it was shown that highly coupled rabbit gamma globulin or Fc fragments are unable to sensitize guinea pig skin for reverse PCA reactions (Hurlimann & Ovary, 1965). For challenge an ahquot of the intact rabbit antibody was used, the same preparation from which the Fc fragment was obtained. In Tables 2 and 3, two representative experiments are presented. The sheep anti-rabbit IgG contained 3 mg antibody directed against the Fc fragment. As can be seen. Fab fragments did not sensitize the skin for reverse PCA reactions whereas the intact antibodies and also the Fc fragments from these antibodies were effective. When 13 groups of DNP were coupled to the Fc fragment no reverse PCA reactions could be produced, but when only 3 DNP groups were coupled to one molecule of Fc fragment good reverse PCA reactions were obtained. This fact was interpreted to indicate that excessive coupling alters the Fc fragment and/or masks the structures necessary for ‘fixation’ to mast cell receptors. Table 3 shows that when challenge was made with the native anti-DNP antibody reverse PCA reactions were obtained only by the DNP,Fc fragment. The rabbit IgG molecule has a structure on its Fc fragment which is recognized by receptors on the mast cell membrane. Receptors for antibody was already demonstrated by Tigelaar ef al. (1971) on mouse mast cells. The Fc fragment from an IgG molecule has two of the C, ‘domains’: Cn2 and C,3 and it was postulated that discrete functions of antibody molecules can be precisely localized to one of the domains (Edelman. 1970). Using the enzyme plasmin the antibody can be separated into two fragments: the Facb fragment, containing the entire IgG molecule less the Cn3 domain and the Cn3 domain itself (Colomb & Porter. 1975). Neither of these two fragments can sensitize for PCA or reverse PCA reactions nor inhibit sensitization with intact antibody for PCA or reverse
754
PCA reactions
Z. OVARY
(Ovary et cd., 1976). In this case at least
the integrity of the together) is necessary.
Fc
fragment
(two
domains
The examples quoted above show that by using PCA and reverse PCA reactions biologic activities of IgG antibodies could be localized with success to more discrete portions of the molecule. Ac,knon,le~/~nrc,rrrs This work was supported by grants from National Institute of Health Grant Al-03075. The National Cancer Institute CA-16247 and the Cancer Research Institute Inc.. New York. The authors express their thanks to Dr. S. Salvatore Caiar~a for his help in the preparation ofthe manuscript.
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14,